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CLINICAL MICROBIOLOGY REVIEWS, Apr. 2007, p. 243267 Vol. 20, No. 20893-8512/07/$08.000 doi:10.1128/CMR.00037-06Copyright 2007, American Society for Microbiology. All Rights Reserved.

Avian Influenza Virus (H5N1): a Threat to Human HealthJ. S. Malik Peiris,1,2* Menno D. de Jong,3 and Yi Guan1

Department of Microbiology, University Pathology Building, Queen Mary Hospital, The University of Hong Kong, Pokfulam,

Hong Kong, SAR, Peoples Republic of China1

; Hong Kong UniversityPasteur Research Centre, Sassoon Rd.,Pokfulam, Hong Kong, SAR, Peoples Republic of China2 ; and Oxford University Clinical Research Unit,

Hospital for Tropical Diseases, 190 Ben Ham Tu, Ho Chi Minh City, Vietnam3

INTRODUCTION ................. ................. ................. .................. ................. ................. .................. .................. ............243THE VIRUS ............... .................. ................. ................. .................. ................. .................. ................. ................. .......244

Biological Properties ..............................................................................................................................................244Ecology of Influenza Viruses .................. ................. ................. .................. ................. .................. ................. .......244Highly Pathogenic Avian Influenza Virus ...........................................................................................................244

BIRD-TO-HUMAN TRANSMISSION OF AVIAN INFLUENZA VIRUSES......................................................245Barriers of Interspecies Transmission.................................................................................................................245Transmission of Avian Influenza Viruses to Humans.......................................................................................246Human Disease Caused by Non-H5 Avian Influenza Viruses ..........................................................................246

ECOLOGY OF AVIAN INFLUENZA A VIRUS (H5N1) ......................................................................................247

Emergence and Spread of Avian Influenza H5N1 Virus...................................................................................247Transmission within Poultry and Wild Birds.....................................................................................................249INFLUENZA H5N1 VIRUS IN HUMANS..............................................................................................................250

Transmission and Epidemiology...........................................................................................................................250The Clinical Spectrum of Human H5N1 Infections...........................................................................................251Pathogenesis of Human Influenza H5N1 Virus..................................................................................................252Laboratory Diagnosis ............... .................. ................. ................. .................. ................. .................. ................. ....254

Clinical specimens for virus detection.............................................................................................................254 Virus isolation .................. ................. ................. .................. ................. ................. .................. ................. ..........255 Antigen detection ................................................................................................................................................255RT-PCR .................. ................. ................. .................. ................. ................. .................. ................. ................. ....255

Antibody detection ................ .................. ................. ................. .................. ................. .................. ................. ....255Biosafety ................ .................. ................. ................. .................. ................. .................. ................. ................. ....256

Antiviral Treatment and Other Options for Therapy .......................................................................................256Infection Control and Prophylaxis ................ ................. ................. .................. ................. .................. ................257

Vaccines....................................................................................................................................................................258OPTIONS FOR INTERVENTION ................. .................. ................. ................. .................. ................. .................. .259

Controlling Transmission in Poultry .................. ................. ................. .................. ................. .................. ..........259Control or Mitigation of a Pandemic...................................................................................................................259

WILL THE NEXT PANDEMIC VIRUS BE H5N1? ..............................................................................................260CONCLUSION............................................................................................................................................................261

ACKNOWLEDGMENTS ...........................................................................................................................................261REFERENCES ................ .................. ................. ................. .................. ................. .................. ................. ................. .261

INTRODUCTION

The highly pathogenic avian influenza (HPAI) H5N1 virus,which is panzootic in poultry, continues to spread and pose amajor challenge to animal and human health (50, 260). Since

pandemic influenza virus has its origins in avian influenza vi-ruses (252), HPAI H5N1 virus has to be considered a poten-tially serious pandemic threat. New influenza virus pandemicsin the 21st century are a certainty, but whether H5N1 will bethe next pandemic virus is far from certain. What is alreadytrue, however, is that H5N1 viruses are taking a huge toll onthe poultry industry in many developing countries, and this

directly or indirectly impacts both economic and social well-

being. The potential impact of HPAI H5N1 virus (and humanreaction to its spread) on wildlife and ecology has received lessattention but is also worthy of consideration (180).

While the H5N1 virus transmits zoonotically from infectedpoultry to humans, often with fatal consequences, such trans-mission remains inefficient. Although the virus replicates effi-ciently in diseased humans, it has not yet adapted to efficient

human-to-human transmission. H5N1 therefore continues to

challenge our understanding of interspecies transmission of

influenza viruses. Here, we review the biology and ecology of

HPAI H5N1 viruses in the broader context of animal and

human influenza viruses in general. We discuss options for the

control of H5N1 transmission in animals and humans and

assess its pandemic risk. We specifically address aspects of

human H5N1 disease in relation to its epidemiology, clinical

presentation, pathogenesis, diagnosis, and management.

* Corresponding author. Mailing address: Department of Microbi-ology, University Pathology Building, Queen Mary Hospital, The Uni-

versity of Hong Kong, Pokfulam, Hong Kong, SAR, Peoples Republicof China. Phone: (852) 2855-4888. Fax: (852) 2855-1241. E-mail: [email protected].

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THE VIRUS

Biological Properties

Influenza A viruses are enveloped RNA viruses with aneight-segmented, single-stranded, negative-sense genome be-longing to the family Orthomyxoviridae. Influenza virus type A

(and type B) causes recurrent epidemics almost every year,leading to significant human morbidity and mortality. How-ever, only influenza A virus is associated with influenza viruspandemics, where an antigenically novel influenza virusemerges to spread rapidly worldwide in an immunologicallyna ve population. In past pandemics, 20 to 30% of the globalpopulation was infected within the first year, and in this regard,influenza A viruses are unique human pathogens. The lastcentury witnessed three such pandemics, in 1918 (the so-calledSpanish flu), 1957 (Asian flu), and 1968 (Hong Kongflu). The pandemic of 1918 is believed to have claimed over 40million lives, while those of 1957 and 1968 are each believed tohave led to over 4 and 1 million deaths, respectively (38, 252).

The eight gene segments of influenza A virus encode 10proteins: hemagglutinin (HA), neuraminidase (NA), matrixproteins M2 and M1, nonstructural (NS) proteins NS1 andNS2, the nucleocapsid, and the three polymerases, the PB1(polymerase basic 1), PB2, and PA (polymerase acidic) pro-teins (252). For some influenza viruses, the PB1 gene hasrecently been discovered to encode an additional protein, thePB1-F2 protein (30). Influenza type A viruses are subtypedbased upon the HA and NA antigens, which are surface pro-teins found on the viral envelope (153). Mutation in thesegenes is selected for by herd-immune selection pressure in thehost, leading to a directional antigenic change over time (an-tigenic drift), thereby explaining the repeated epidemics ob-served with influenza A or B virus. The segmented genome ofinfluenza viruses also allows for genetic reassortment to occurwhen two influenza viruses infect the same cell (18, 142). Thisprovides influenza viruses a powerful option for the generationof genetic diversity for interspecies transmission and to evadehost immune responses through a major antigenic change(antigenic shift). Pandemics arise at infrequent intervals when an influenza virus with a completely novel HA (andsometimes NA) acquires the ability for efficient and sustainedhuman-to-human transmission in a population that is immu-nologically na ve to the virus surface proteins (HA and NA).The H2N2 influenza virus responsible for the pandemic of1957 arose through genetic reassortment, where the prevailinghuman influenza A virus (H1N1) acquired the HA (H2), NA(N2), and PB1 genes from an avian virus (101, 120, 187, 252).Similarly, the pandemic of 1968 arose through the acquisitionof a novel HA (H3) and the PB1 gene from an avian source (6,101). In contrast, the pandemic of 1918 is believed to havearisen through the direct adaptation of a purely avian virus toefficient transmission in humans (226), although the lack ofgenetic information on relevant avian precursors and on thepre-1918 human viruses precludes a definitive conclusion onthis matter (62). Thus, pandemic influenza virus is a zoonosis,and avian viruses play a critical role in its genesis (194). Sincethe pandemics of 1957 and 1968 arose in southern China, thisregion has been identified as a hypothetical pandemic epicen-ter (196).

Ecology of Influenza Viruses

Sixteen subtypes of HA (H1 to H16) and nine subtypes of NA(N1 to N9) are recognized in aquatic birds (53, 252). While manyof these subtypes can be consistently detected in wild aquatic

waterfowl, only few subtypes have established themselves in mam-malian species such as humans (HA [H1, H2, and H3] and NA[N1 and N2]), pigs (HA [H1 and H3] and NA [N1 and N2]),horses (H3N8 and H7N7), and dogs (H3N8) (Fig. 1). Indeed,only some of the diverse influenza virus subtypes found in aquaticbirds have established themselves as low-pathogenicity avian in-fluenza (LPAI) virus in terrestrial poultry such as chicken, turkey,and quail (e.g., subtypes H9 and H6) (see below).

Both human and avian influenza viruses have establishedstable virus lineages in pigs, possibly a reflection of the fact thatreceptors for both avian and human influenza viruses arepresent on the porcine epithelium (90). For these reasons, pigshave been regarded as being a possible intermediate host

(mixing vessel) for the generation of pandemic influenza virus through reassortment (139). Human influenza virusesthat have become established in pigs include classical swineH1N1 and H3N2 viruses and reassortants thereof (H1N2 andH3N1) (164, 169, 188, 197). The 1918 H1N1 virus appears tohave entered human and pig populations, although the epide-miological evidence favors the initial host as being humans(225). Avian-like H1N1 viruses have established themselves inpigs in Europe (139, 188). In addition, other viruses have beentransiently detected in pig populations. These include avianvirus subtypes H1N1 (Asia), H4N6 (Canada), H9N2 (China),and H5N1 (Asia) (33, 71, 164, 169).

More recently, equine H3N8 viruses have been transmitted toracing dog populations in the United States, possibly facilitated bythe practice of feeding horsemeat to racing dogs, another exam-ple of a human intervention that promoted interspecies transmis-sion of viruses (37). Overall, there are strong barriers to interspe-cies transmission that prevent the adaptation of influenza virusesto new hosts (see below). It is likely that these prevent the morefrequent emergence of pandemics from the wide diversity of HAsubtypes prevalent in waterfowl.

Highly Pathogenic Avian Influenza Virus

Two subtypes of influenza A virus (H5 and H7) are known togive rise to HPAI virus in terrestrial poultry (chicken andturkeys). The HPAI virus phenotypes of these viruses are re-

FIG. 1. Ecology of influenza A viruses and interspecies transmission.

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lated largely, though not exclusively, to mutations giving rise tomultiple basic amino acids in the connecting peptide betweenthe HA1 and HA2 domains of the HA0 precursor protein (84,109). In the viral life cycle, posttranslational cleavage of theprecursor HA molecule (HA0) into two subunits (HA1 andHA2) by host proteases is essential for productive virus repli-cation, since this generates a fusogenic domain mediating thefusion between the viral envelope and the endosomal mem-brane. This may occur extracellularly by trypsin-like proteasesthat are restricted in tissue distribution to the respiratory andgastrointestinal tracts. However, when multiple basic aminoacids are introduced into the HA cleavage site, the HA0 pre-cursor becomes cleavable by a wide range of proteases (e.g.,furins [PC6-like]) with ubiquitous tissue distribution (105,246). This permits productive virus replication in organs out-side the respiratory and gastrointestinal tracts, including thebrain, resulting in fulminant disseminated disease with highmortality, leading to HPAI virus (84). The acquisition of acarbohydrate side chain near the cleavage site can modulatethe pathogenicity of a virus by masking the accessibility of the

proteases to the cleavage site (102, 84).In the 31 years from 1959 to 1990, there were nine HPAI

virus outbreaks recorded in Europe, North America, and Aus-tralia, and these outbreaks were contained by the stampingout of infected flocks (1). In the 11 years since 1990, therehave been 10 further HPAI virus outbreaks, including in Asia.The current HPAI H5N1 virus outbreak (from 2003 onwards)is, however, unprecedented in scale and geographic distribu-tion. These viruses are now panzootic across three continents,leading to huge economic losses, and have transmitted to hu-mans with lethal consequences. The expansion of intensivepoultry husbandry, which is the fastest growing livestock indus-try globally, with an estimated 16 billion chickens and 1 billion

ducks worldwide, is likely facilitating the increasing frequencyand scale of HPAI virus outbreaks. Furthermore, the commer-cialized large-scale poultry industry is now associated with themovement of live poultry and poultry products over long dis-tances, thereby facilitating the transmission of infection.

On the basis of the genetic sequence of HA and the biolog-ical properties of the virus, it appears that the avian influenzaviruses that contributed to the origin of the pandemics of 1957and 1968 were LPAI viruses of chicken and other terrestrialpoultry. Therefore, for pandemic preparedness, surveillance ofpoultry and other avian species must be directed at healthy aswell as diseased birds. On the other hand, reconstruction of theH1N1 virus causing the Spanish flu pandemic of 1918 sug-gests that this virus may have had high pathogenicity for ter-restrial poultry even though it did not have the multibasiccleavage site in the HA that characterizes HPAI virus (236).However, direct proof of high pathogenicity of the 1918 virusfor chickens is still awaited.

BIRD-TO-HUMAN TRANSMISSION OF AVIAN

INFLUENZA VIRUSES

Barriers of Interspecies Transmission

Avian influenza viruses do not efficiently infect humans ornonhuman primates (7, 152). Conversely, human viruses donot efficiently replicate in ducks (78). The viral and host factors

that determine host restriction are poorly understood (83, 84,116, 156, 249) and are believed to be determined by multiple viral genetic determinants including the viral HA and NAgenes as well as other internal genes such as the nucleoproteinand the PB2 genes.

The HA of human influenza viruses binds to cell sialic acidlinked to galactose by an -2,6 linkage (SA -2,6) found on

human cells, while avian viruses have a predilection for sialicacid linked to galactose by -2,3 linkages (SA -2,3) found onavian (e.g., duck) epithelia (181). This receptor specificity wasbelieved to be one of the factors responsible for the speciesbarrier that keeps avian viruses from readily infecting humans.More recently, it has been shown that the epithelial cells of thelower respiratory tract (viz., terminal bronchioles and alveolarepithelial cells) have both SA -2,3 and SA -2,6 receptors.Furthermore, fluorescein-labeled virus was shown to bind ef-ficiently to epithelial cells of the terminal bronchioles andalveoli, and avian-like H5N1 viruses were shown to infect andreplicate in ex vivo cultures of human lung fragments (159, 192,243). Although the putative SA -2,3 receptors for H5N1 vi-

ruses appear to be lacking in the upper respiratory tract, H5N1 viruses are able to replicate in ex vivo organ cultures of theupper respiratory tract (159). On the other hand, some H5N1viruses isolated from humans appear to have acquired muta-tions in HA associated with a change in affinity from SA -2,3and SA -2,6 receptors (263), although such mutations werenot by themselves sufficient for these viruses to be efficientlytransmitted from human to human. Thus, the paradigm of SA-2,3 and SA -2,6 receptors in explaining the tissue tropismand host restriction of avian influenza viruses probably de-serves to be reassessed. Novel technologies such as glycanmicroarrays are revealing that the situation is indeed complex,with different virus strains binding to novel structures such as

sulfated and sialylated glycans in addition to the conventionalSA -2,3 and SA -2,6 (57, 212, 213). Additional informationabout the glycans on the surface of host cells combined withdata on the predilections of virus binding to these structuresare likely to provide new biological insights with regard tointerspecies transmission of avian influenza viruses.

A layer of mucin covers the respiratory epithelium, forminga barrier that the virus has to penetrate before it can attach tothe cell surface (191). This also plays an important role in hostsusceptibility and tissue tropism. Mucin is rich in sialic acidsthat act as decoys, trapping the virus and preventing it fromreaching the cell surface (5, 147). By cleaving sialic acids inmucus that would otherwise impede the spread of the virus,NA promotes its spread within the respiratory tract.

The NA of avian influenza viruses has a predilection for thehydrolyzation of the avian SA -2,3 Gal linkage rather than themammalian SA -2,6 linkage. A balance between the activityof HA in virus attachment and NA in virus release needs to bemaintained for optimal viral replication (245). Deletions in thestalk of the NA reduces the effective enzymatic activity of theNA (12). The H5N1 viruses that have adapted to terrestrialpoultry (e.g., chicken) in Hong Kong in 1997 and more recently(2002 to present) have deletions in the stalk region of NA(127), which perhaps allows the virus NA activity to balancethe weaker interaction of the H5 HA with the viral receptor ofchicken compared to that of aquatic birds.

Avian influenza viruses typically have Glu627 in the PB2

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gene, while human viruses have Lys627. This mutation may beassociated with temperature sensitivity (146) and seems to beinvolved in host adaptation (217), possibly by playing a role inadaptation to replication and transmission within mammals.The role of PB2 in the pathogenesis of H5N1 viruses is dealtwith below (see Pathogenesis of Human Influenza H5N1 Vi-ruses). During the emergence of the pandemic viruses of 1957

and 1968, in addition to the novel HA (and also, in 1957, NA),the PB1 gene was also acquired from the avian influenza virusprecursor (101). In minireplicon systems investigating the com-patibility of avian and human polymerase components, anavian PB1 appeared to provide a replicative advantage inmammalian cells (154). However, in other studies, human virusreassortants carrying avian PB1 appeared to have reducedreplication competence (204). Thus, the role of avian PB1 inthe emergence of pandemic influenza virus reassortants stillremains unclear. The importance of the polymerase complex inthe adaptation to new hosts is being increasingly recognizedand is discussed further below (55, 184).

Transmission of Avian Influenza Viruses to Humans

The ability of avian influenza A viruses of subtypes H1N1,H3N8, H3N2, H6N2, H6N1, H9N2, H4N8, and H10N7 toreplicate in humans was investigated by experimental infectionof 81 healthy human volunteers (7). Some volunteers experi-mentally infected with H4N8, H10N7, or H6N1 virus had ev-idence of viral replication in the nasopharynx, and some hadmild upper respiratory symptoms. None of them had evidenceof rising antibody titers using the conventional hemagglutina-tion inhibition (HI) test. Neutralizing antibody responses werenot assessed. Attempts to artificially transfer H6N1 from onevolunteer to another were unsuccessful. Volunteers infectedwith avian H1N1, H3N2, H3N8, H6N2, or H9N2 virus had noevidence of virus replication in the nasopharynx, but some ofthem had serological responses to the infecting virus. It wasspeculated that natural infections by human viruses of subtypesH1 or H3 and N1 or N2 may have provided cross-reactingimmunity that prevented avian virus replication. These findingshighlight the fact that avian influenza viruses can infect hu-mans, at least following experimental challenge, and also thatconventional HI tests underestimate such infections.

Human sera collected in southern China from the late 1970sto the early 1980s had evidence of antibodies to a number ofLPAI virus subtypes (e.g., H4, H5, H6, H7, H10, and H11), asassessed by the single radial hemolysis test (194) (K. F. Shor-tridge, personal communication). Human seroprevalence ap-

parently correlated with the isolation rates of viruses in ducks, with some exceptions (e.g., H7). The seroprevalence for H5viruses ranged from 0% (Hong Kong) to 2.3% (Jiangsu Prov-ince). It should be noted that this H5 seroprevalence likelyreflects exposure to low-pathogenicity H5 viruses present inducks; it does not provide evidence of continued exposure tothe current HPAI H5N1 virus.

Human Disease Caused by Non-H5 Avian Influenza Viruses

Besides H5N1, the direct transmission of avian influenzaviruses of subtypes H7N7, H9N2, and H7N3 has been associ-ated with human disease (Table 1). It is notable that the virussubtypes causing zoonotic human disease have been largelythose known to cause infection in terrestrial poultry. It is un-clear whether this reflects the increased probability of an ex-posure event (chickens being more abundant than ducks andhuman exposure to poultry being more common than exposureto aquatic wild birds) or whether viruses that have adapted toterrestrial poultry have a greater propensity to transmit to

humans. Of note, terrestrial poultry such as chicken and quailhave SA -2,6 receptors that bind human influenza viruses (56,247). This may explain the observation that H9N2 viruses,which are endemic in quail and chickens in Asia, have a pre-dilection to bind to both human SA -2,6 and avian SA -2,3receptors (148). It may also explain why only a smaller subset(e.g., H9 and H6) of the diverse HA subtypes present inaquatic birds has become endemic in terrestrial poultry. Itraises the possibility that terrestrial poultry may facilitate theadaptation of avian influenza virus to more efficient binding tothe human SA -2,6 receptors.

H7N7 virus was isolated from the blood clot of a man withclinical features compatible with a hepatitis-like illness, but therelationship of this isolate to the disease is unclear (22, 42).There are also reports of self-limited conjunctivitis caused byH7N7 viruses after accidental infection in the laboratory, afterinfection of an animal (seal) handler, and from backyard poul-try (4, 119, 227, 253). During the large-scale 2003 outbreak ofH7N7 virus in Dutch poultry, active case finding among ex-posed persons and their close contacts identified a total of 89laboratory-confirmed infections in humans (112), amountingto approximately 2% of the estimated number of people po-tentially exposed to the virus. The highest infection rates wereobserved in veterinarians and persons involved in the culling ofchickens. During the same outbreak, human-to-human trans-mission of H7N7 virus was suggested for three individuals whohad not been in direct contact with infected poultry but were

TABLE 1. Human disease caused by interspecies transmission of avian influenza viruses without prior reassortment

Yr Antigenic description and interspecies transmission event

Pre-1997.................................................................H1N1, 1918 Spanish flu pandemic?H7N7, sporadic conjunctivitis

1997........................................................................H5N1 in Hong Kong, 18 cases and 6 deaths1998, 1999, 2003 ...................................................H9N2 in Hong Kong and Guangdong2003........................................................................H5N1 diagnosed in Hong Kong (likely acquired in Fujian Province, Peoples Republic of China)

and in mainland China2003........................................................................H7N7 in Holland; 78 cases of conjunctivitis, 7 with flu-like illness, 4 others, 1 death2004 ........................................................................H7N3 in Canada; conjunctivitis2004present .........................................................H5N1 disease and death in southeast and east Asia, central Asia, the Middle East, and Africa


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family members of poultry workers with symptomatic H7N7infections (112). Most of these patients (83 of 85) presentedwith conjunctivitis, while seven patients presented with a febrileflu-like illness (54, 112). Five patients with conjunctivitis alsohad a flu-like illness. A veterinarian who had visited an affectedfarm subsequently developed pneumonia complicated by acuterespiratory distress syndrome (ARDS) and multiorgan failureand succumbed to his illness (54).

An outbreak of HPAI H7N3 virus in poultry farms in BritishColumbia, Canada, led to self-limited conjunctivitis and flu-like illness in two people exposed to infected poultry (79, 239).H7N3 virus was isolated from both patients. Poultry workersexposed to poultry affected by LPAI and HPAI virus outbreaksof H7N3 virus in Italy had evidence of H7 seropositivity in3.8% of those tested (176).

It is notable that H7-subtype influenza viruses have a tro-pism for the conjunctiva. Viral load was higher in the conjunc-tival swabs than in respiratory specimens, supporting this con-tention (54). It was reported that the conjunctiva contains apredominance of SA -2,3 receptors, which may explain this

tropism of the virus (163). Interestingly, other avian viruses(e.g., H5N1), which also have an SA -2,3 binding preference,do not appear to cause conjunctivitis, although the conjunctivamay conceivably provide a portal of entry for the virus.

Avian (low-pathogenicity) H9N2 viruses have repeatedly in-fected humans in Hong Kong and in Guangdong, giving rise toa mild flu-like illness (19, 74, 131, 171). These virus strainsbelonged to the A/Duck/Hong Kong/Y280/97-like or theA/Quail/Hong Kong/G1/97-like virus lineages. Although H9N2 viruses bind human SA -2,6 receptors (148), no human-to-human transmission was detected.

ECOLOGY OF AVIAN INFLUENZA A VIRUS (H5N1)

Emergence and Spread of Avian Influenza H5N1 Virus

The HPAI H5N1 virus lineage currently endemic in Asiawas first detected in diseased geese in Guangdong Province,Peoples Republic of China, and designated A/Goose/Guangdong/1/96 (262). The H5N1 bird flu incident in HongKong in 1997 was the first known instance of a purely avian virus causing severe human disease and death, with 18 hu-man cases, 6 of whom died (36, 218, 266). The slaughter ofall (1.5 million) poultry in the farms and markets of HongKong aborted this outbreak. This virus (H5N1/97) was infact a reassortant virus with the HA (H5) being derived from A/Goose/Guangdong/1/96 and the other genes being de-rived from H9N2 and H6N1 viruses prevalent in quail (72,82, 262). The viral NA had a deletion in the stalk region ofthe NA, which is associated with the adaptation of influenza viruses to terrestrial poultry such as chickens. While theH5N1/97 virus was not seen since the poultry slaughter ofDecember 1997, continued surveillance of poultry in HongKong revealed the presence of A/Goose/Guangdong/1/96-like viruses in imported geese (68). From 2000 onwards, aseries of reassortants were detected in ducks, chicken, andother terrestrial poultry, with the HA being derived fromthe A/Goose/Guandong/1/96-like lineage but with the inter-nal gene segments being derived from other influenza vi-ruses from birds (Fig. 2) (67, 69). A diversity of genotypes (a

genotype is a designation that reflects the constellation ofeight gene segments of the virus) was seen in 2001 and 2002,but by 2003, genotype Z began to emerge as a dominantgenotype in terrestrial poultry in southern China. It hadagain acquired a deletion in the stalk region of the NA (notidentical but overlapping that observed in H5N1 viruses inHong Kong in 1997) that characterizes adaptation to terres-trial poultry. HPAI H5N1 virus was also documented inducks in mainland China since 1999 (26), in Geese in Viet-nam during 2001 (158), and in imported duck meat fromChina in 2001 (138, 238), indicating that these viruses con-tinued to circulate in ducks and geese in the region. Inaddition, in 2002, HPAI H5N1 virus was documented in

FIG. 2. Genetic reassortment and emergence of influenza A virus(H5N1) in Asia from 1999 to 2005. The eight gene segments (repre-sented by horizontal bars) starting from top downwards are PB2, PB1,PA, HA, NP, NA, M, and NS. Each color represents a distinct viruslineage. Red represents the A/Goose/Guangong/1/96-like lineage. Dis-tinct genotypes (i.e., gene constellations) are denoted by a letter. Theorigin of the 1997 virus in Hong Kong is not represented here but wasa separate reassortment event with only the HA being derived from

A/Goose/Guangdong/1/96 and the other seven gene segments beingderived from H9N2 and H6N1 avian influenza viruses found in quail.(Adapted from reference 127 by permission from Macmillan Publish-ers Ltd.)


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dead wild birds in Hong Kong (46, 69). Some of these viruses, especially those of genotype Z, acquired pathoge-nicity for aquatic birds such as ducks and led to severeneurological disease (46). This was unusual, since evenHPAI viruses are usually nonlethal for ducks.

From December 2003 onward, Japan, South Korea, Viet-nam, Thailand, Indonesia, mainland China, Cambodia, Laos,and Malaysia reported outbreaks of HPAI H5N1 virus disease(http://gamapserver.who.int/mapLibrary/app/searchResults.aspx)(199). The viruses introduced into Japan and South Korea

were genotype V viruses, but those that became dominant inVietnam, Thailand, Cambodia, Indonesia, and southern Chinawere the Z genotype virus, which first emerged in 2002 and hassince become the dominant virus genotype in terrestrial poul-try (Fig. 2) (127). In 2004 and early 2005, H5N1 viruses in Asiarevealed significant genetic and antigenic heterogeneity (2, 28,203). Viruses in mainland China were diverse and comprisedmultiple geographically related sublineages of multiple geno-types (28).

In contrast, viruses found in Vietnam, Thailand, Cambo-dia, and Malaysia (Z genotype) formed one closely relatedclade (clade 1), while those found in Indonesia (Z genotype)formed another group of closely related viruses (clade 2.1).This suggested that the viruses responsible for the poultryoutbreaks in Indochina on the one hand and in Indonesia onthe other were each common-source introductions and thatthe perpetuation of H5N1 viruses in Indochina and Indone-sia, respectively, were until recently maintained within poul-try rather than through the repeated reintroduction of vi-ruses from external sources. However, more recentlyisolated H5N1 viruses from Vietnam suggest that geneticallydistinct viruses are now being introduced, likely via legal orillegal poultry trade (203).

In May 2005, an outbreak of H5N1 led to the death of over6,000 migratory waterfowl in Qinghai Lake in western China(27, 29, 134). This is a major breeding site for migratory birdswhose flyways extend to India, Siberia, and southeast Asia. The

species affected were bar-headed geese (Anser indicus), black-headed gulls ( Larus ichthyaetus), brown-headed gulls (Larusbrunnicephalus), ruddy shelducks (Tadorna ferruginea), and alimited number of great cormorants ( Phalacrocorax carbo).Smaller numbers of whooper swans, black-headed cranes, andpochards were also affected. It is unclear whether the virus wasbrought to Qinghai by bird migration or whether it was ac-quired locally from infected poultry. However, this was the firstsustained major outbreak within wild bird populations to bedocumented. Subsequently, H5N1 outbreaks in wild birds or in

poultry were reported in Siberia (July 2005), Mongolia andKazakhstan (August 2005), Romania, Croatia, and Turkey(October 2005). Wild bird infections with or without poultrydisease were noted in a number of other Middle Easternand European countries in 2006. Infection was detected inpoultry flocks in Nigeria and India in February 2006. Re-currences of wild bird outbreaks affecting similar bird spe-cies occurred in Liaoning and Qinghai, Peoples Republicof China, in April 2006 (http://www.fao.org/docs/eims/upload//211696/EW_asia_August06.pdf).

Surveillance carried out in six provinces of southern Chinafrom July 2005 to June 2006 revealed that the geographicallydistinct H5 sublineages documented in previous years (seeabove) (28) had been replaced largely by one dominant sub-lineage (clade 2.3) (202). Viruses isolated recently from humancases from China also belonged to this same sublineage, as didH5N1 viruses isolated in 2006 from poultry in Malaysia andLaos and from wild birds in Hong Kong. This suggests theemergence and predominance of this sublineage in southernChina and southeast Asia (202). Comparable data from otherparts of China and Asia are lacking.

By July 2006, 54 countries across three continents had beenaffected by this H5N1 panzootic virus, and transmission tohumans and to other species has been reported in some ofthese countries (Table 2) (http://gamapserver.who.int/mapLibrary/app/searchResults.aspx). Over 209 million poultry have died orbeen culled since January 2004 (50).

TABLE 2. Cumulative number of confirmed human cases of avian influenza A virus (H5N1) reported to the WHO as of 3 February 2007a

CountryDate of report

of first case

No.

2003 2004 2005 2006 2007b Totalb

Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths

Vietnam 11 January 2004c 3 3 29 20 61 19 0 0 0 0 93 42Thailand 23 January 2004 0 0 17 12 5 2 3 3 0 0 25 17Cambodia 2 February 2005 0 0 0 0 4 4 2 2 0 0 6 6Indonesia 21 July 2005 0 0 0 0 19 12 56 46 6 5 81 63China 19 November 2005d 1 1 0 0 8 5 13 8 0 0 22 14Turkey 5 January 2006 0 0 0 0 0 0 12 4 0 0 12 4Iraq 30 January 2006 0 0 0 0 0 0 3 2 0 0 3 2

Azerbaijan 14 March 2006 0 0 0 0 0 0 8 5 0 0 8 5Egypt 20 March 2006 0 0 0 0 0 0 18 10 1 1 19 11Djibouti 12 May 2006 0 0 0 0 0 0 1 0 0 0 1 0Nigeria 3 February 2007 0 0 0 0 0 0 0 0 1 1 1 1

Total 4 4 46 32 97 42 116 80 8 7 271 165

a Data are adapted from the WHO website (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_02_06/en/index.html).b Up to 3 Feb 2007.c Date of first case represents data of reporting to the WHO. In some instances, the onset of the case predates the date of reporting considerably.d Two patients diagnosed in Hong Kong in February 2003 who likely acquired infection through travel in Fujian Province, Peoples Republic of China, in January

and February 2003 (170) are not included in this table.


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Transmission within Poultry and Wild Birds

From 2003 to 2004, those Asian countries that detected theintroduction of H5N1 virus infection into their poultry flocksearly (e.g., Japan, South Korea, and Malaysia) were successfulin rapidly eradicating the infection by aggressive stamping-out policies. However, once the virus becomes entrenched, it

becomes extremely difficult to eradicate (199, 229). The rea-sons for this are multiple but include a high prevalence ofbackyard flocks, mixed chicken and duck rearing, asymptom-atic virus shedding in ducks, live-poultry markets, and legal andillegal wildlife trade (141, 86). A high proportion of villagehouseholds in Asia have backyard poultry, with many of themhaving chickens and ducks as well as pigs in close proximity(141). Almost 70% of poultry production in Asia is in backyardflocks, where biosecurity is impossible to implement and whichis associated with poor access to veterinary services, leading tononrecognition or delayed recognition of disease outbreaks.The lack of effective compensation schemes for affected flocksmay lead to the underreporting of disease.

Live-poultry markets, especially those permanent marketswhere unsold poultry are held overnight within the market, areknown to amplify and maintain avian influenza viruses withinthem. Virus isolation rates in these live-poultry markets aregenerally higher than those detected in incoming poultry, andthus, these markets are an ideal site for avian influenza virussurveillance (J. S. M. Peiris and Y. Guan, unpublished data).Once viruses enter such a market with incoming poultry, theycontinue to circulate and amplify within the poultry market.Virus transmission can be interrupted only by a rest day,when the market is completely emptied of poultry (118). Suchsurveillance studies in southern China have documented sig-nificant HPAI H5N1 virus isolation rates from apparently

healthy birds, especially in ducks and geese (28, 127). Virusisolation rates increase in the cooler winter months (28, 127).Furthermore, while live-poultry markets are a dead end forpoultry (which end up being slaughtered), they are not deadends for virus transmission. There is evidence that in additionto amplifying and perpetuating virus within them, these poultrymarkets serve as a source of infection of poultry farms via themovement of empty cages and personnel (117). This is perhapsaccentuated by the fact that small-holder poultry farmerswhose flocks have developed H5N1 disease try to salvage someof their investment by selling the surviving chickens to suchpoultry markets and slaughterhouses. In village-level studies ofdisease in backyard flocks, recent purchase of poultry was arisk factor for introduction of disease (244). In summary, thedata from Hong Kong and southeast Asia indicate that farfrom being dead ends in the transmission cycle, these live-poultry markets are a key factor in the maintenance of virus inthe poultry marketing system. It is unclear whether these mar-kets play a similar role in other Asian countries such as Viet-nam and Indonesia. If so, it provides one option for interven-tion to interrupt virus transmission.

Ducks may be infected with H5N1 HPAI viruses withoutshowing overt signs of illness and continue to shed virus for upto 17 days (86, 214, 215). While replicating within a duck,H5N1 viruses appear to lose virulence for ducks and alsoundergo an antigenic change, which allows the virus to persistand reinfect in the face of a serological response to the initial

virus (86). Although such viruses are less virulent for ducks,they retain virulence for chickens and remain HPAI viruses. Insome regions of Asia, ducks are led to graze on harvested ricefields and may be moved large distances over time. Thus,infected free-range ducks may serve as Trojan horses, intro-ducing virus into local farms and backyard flocks within unsus-pecting villages. Studies in Thailand have demonstrated that an

abundance of free-grazing ducks is a risk factor for the persis-tence and spread of HPAI virus (63, 206).

Other factors that may help to spread HPAI virus includefighting cocks that are moved from place to place, even acrosscountry borders, for cockfights. A well-organized trade in poul-try, poultry products, and other birds (e.g., pet birds) withincountries and also (often illegally) across borders promotes thedissemination of the virus. Birds exported as a part of theextensive trade in pet birds have also been occasionally foundto be infected with HPAI H5N1 virus (199, 242). Religiouspractices that foster large-scale bird release in some Asiancountries have led to a major trade in passerine birds (R. T.Corlett, personal communication), and this may provide an

additional route for the spread of avian influenza viruses. Fur-thermore, poultry feces are often used as fertilizer or for feed-ing fish, and their movement provides an opportunity for thedissemination of infection. Vaccination of poultry without ad-equate safeguards (e.g., unvaccinated sentinels and serologicalsurveillance to differentiate infected from vaccinated birds)(23, 221) to detect low-level virus circulation within poultryflocks may itself contribute to increasing the likelihood of thesilent spread of H5N1 in vaccinated poultry (186).

A range of influenza viruses are endemic in wild birds, par-ticularly aquatic waterfowl. In past HPAI virus outbreaks inpoultry, it was believed that a precursor low-pathogenicity H5-or H7-subtype virus was introduced into domestic poultry

flocks and thereby acquired mutations in the HA connectingpeptide that conferred high pathogenicity for poultry (91).Since 2002, a range of feral bird species has been affected byHPAI H5N1 virus (46, 113, 145). What remained unclear untilthe Qinghai Lake outbreak in 2005 (see above) (29) was whether the HPAI H5N1 virus can maintain transmissionwithin such wild bird and migrating bird populations.

In contrast to the great genetic diversity of H5N1 viruses inChina and other parts of Asia (28), those viruses causing dis-ease in wild birds or poultry in Mongolia, Siberia, Iraq, Turkey,Central Europe, Germany, Italy, Nigeria, Egypt, and India allwere phylogenetically closely related to each other and to theQinghai-like lineage (clade 2.2) of viruses (45, 168, 251). Giventhe great genetic diversity of H5N1 viruses in Asia (28), it isstriking that only a narrow spectrum of this genetic diversity(i.e., the Qinghai-like clade 2.2) has contributed to the recentspread westward to Europe and southward to India (45, 168,251). Among 390 H5N1 viruses isolated during prospectivesurveillance of poultry markets in six provinces in southernChina carried out from July 2005 to June 2006, only one Qing-hai-like (clade 2.2) virus was identified (202), although datafrom other parts of China are lacking. If the movement ofpoultry or poultry products was responsible for introducingH5N1 into these diverse central Asian, European, African, andIndian locations, it is difficult to explain why only one sublin-eage was responsible for this rapid expansion in geographicextent and why it happens to be the same sublineage respon-


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sible for the largest outbreak of disease in wild birds in QinghaiLake.

It should be noted that the bird species that manifest mor-tality may not necessarily be the species that carry the virusover long distances. While the movement of poultry and poultryproducts cannot be completely excluded as contributing to thisrecent spread of the H5N1 virus, the genetic evidence, takentogether with the epidemiological evidence, is suggestive thatthis geographic expansion of the virus in 2005 was contributed

to largely by bird migration likely amplified by local or regionalmovement of poultry products. An integrated analysis of phy-logenetics, migratory bird movements, and poultry trade hassuggested that the movement of poultry and poultry productsmay have been the dominant vector in the outbreak of H5N1infection during 2003 and 2004 in Asia and its continued en-demicity but that migrating birds appear likely to be implicatedin the westward spread of the virus to Europe and the MiddleEast from 2003 to 2006 (107).

It is still unclear, however, whether wild migrating birds nowprovide a reservoir for HPAI H5N1 virus (50). This distinctionis fundamental for control strategies. If wild birds invariablyacquire the virus from infected poultry, this implies that thecontrol of poultry infection will lead to the eradication of the virus. If, on the other hand, wild birds are one reservoir forHPAI H5N1 virus, even the eradication of poultry infectionwill not lead to the eradication of this pathogenic virus, and itwill continue to seed new poultry outbreaks. The reemergenceof H5N1 in Europe (Hungary) in January 2007 and the pre-liminary reports that the virus in Hungary has high genetichomology to those found in Europe in early 2006 may indicatethat the Qinghai-like clade 2.2 viruses may indeed have be-come entrenched in wild bird populations. (http://www.promedmail.org/pls/promed/ [archive no. 20070130.0389]).However, a more detailed analysis is awaited. The bird speciesfound to be infected with H5N1 have been summarized (107,180).

The recently isolated H5N1 viruses have shown the ability tocross species barriers and infect a range of mammalian speciesincluding humans. Tigers, leopards, and cats have been in-fected by H5N1 virus by feeding on infected poultry or birdcarcasses (103, 205, 265). Owstens civets were infected in ananimal rescue center in Vietnam, although these animals werenot fed infected poultry products (180). In these instances, virus disseminated to involve multiple organs including thebrain. There is serological evidence of occasional infection of

pigs by H5N1 virus, although the virus does not appear to havebecome endemic in pigs (33). Experimental inoculation of pigshas led to infection but has not led to the transmission ofinfection from pig to pig (33, 89). Overall, the possible role ofmammals as intermediate hosts in the transmission of H5N1virus to humans is still poorly explored (115). This is particu-larly relevant since some patients with H5N1 disease have noobvious exposure to poultry (97, 151). Evidence of subclinicalinfection of cats exposed to infected birds (126) and reports ofcats dying of H5N1 disease in a household in Iraq affected bypoultry deaths (265) highlight the possibility that such second-ary vectors may be potentially important as a source of infec-tion of humans. Current knowledge of the ecology of the cur-rent H5N1 outbreak is summarized in Fig. 3.

INFLUENZA H5N1 VIRUS IN HUMANS

Transmission and Epidemiology

The first human disease caused by H5N1 was reported inHong Kong in 1997, with 18 cases and six deaths (see above)(36, 218, 266). The source of human infection appeared to belive-poultry markets where chickens, ducks, geese, and otherspecies of minor poultry (e.g., quail, pheasant, chukka, pigeon,etc.) were sold for human consumption (151, 195). In February2003, as the world was girding itself to confront severe acuterespiratory syndrome, H5N1 disease was diagnosed in Hong

FIG. 3. Ecology, maintenance, and spread of H5N1 viruses (based on data from references 27, 33, 46, 107, 126, 179, 180, 202, 205, and 265).


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Kong in a father and son who had just returned from a holidayin Fujian Province, Peoples Republic of China (170). Thesetwo patients were infected by a genotype Z virus, except that itdid not have a deletion in the stalk region of the NA (desig-nated genotype Z) (69). This virus had an amino acid re-placement at position 227 (H3 numbering) in the receptorbinding pocket of the HA that changed its receptor bindingprofile to recognize both the avian SA -2,3 as well as thehuman SA -2,6 receptors (193). However, by itself, this didnot appear to change its capacity for human-to-human trans-mission. In retrospect, another case of H5N1 occurred in Bei- jing, Peoples Republic of China, in November 2003 (268).Subsequently, with the increasing spread of H5N1 disease inpoultry, further human cases from Vietnam, Thailand, Cam-bodia, Indonesia, and elsewhere were reported (Table 2). In anumber of instances, the detection of a human case in a regionwas the first indication of the presence of poultry infection inthat locality. Taken together, the human cases appear to in-crease in the winter and spring months (260). This correlates with the seasonality observed in virus detection in poultry

(28, 127).Since HPAI H5N1 virus in poultry is associated with the

presence of infectious virus in many organs, as well as theexcretion of large amounts of virus in the feces and othersecretions, sick poultry are a major source of human infection(138, 222). Most human cases of H5N1 infection were associ-ated with the direct handling of infected poultry, slaughteringor preparing sick poultry for consumption, consumption ofuncooked poultry products such as raw blood, or close contactwith live poultry (8, 34, 44, 151, 232, 259). Since H5N1 infec-tion may not always be overtly symptomatic, especially so inducks, even asymptomatic poultry may pose an infection risk,e.g., at wet markets, in areas of endemicity (127, 215). Contact

with a contaminated environment, such as water and poultryfeces used as fertilizer or fish feed, has been suspected to be asource of infection in human H5N1 cases who had no directexposure to poultry (39, 97). In bird-to-human transmission,the likely portal of virus entry is via the respiratory tract, thegastrointestinal tract, or the conjunctiva. Cats experimentallyinfected with H5N1 virus after feeding on infected chickensshowed evidence of viral replication in gastrointestinal plexi.However, this is not seen in those infected via the respiratoryroute (179). In humans, the possibility of intestinal infection issupported by reports of H5N1-infected patients who presentedwith diarrhea as the only initial symptom as well as by patientswho reported consumption of raw duck blood as the sole ex-posure to poultry (3, 8, 39). In addition, the presence of infec-tious virus in fecal material may indicate virus replication inthe human gastrointestinal tract (39, 40, 240).

There are a number of enigmas with regard to human H5N1infection and disease. In spite of large-scale outbreaks ofH5N1 viruses among poultry in densely populated areas andpresumably massive exposure of humans to the virus, the num-ber of reported H5N1 patients has so far been relatively small(244). In Hong Kong in 1997, where there was excellent sur-veillance for symptomatic influenza virus, there were still onlysmall numbers of cases in spite of the exceedingly heavy virusload in retail poultry markets, where 20% of poultry wereinfected (195). Seroepidemiological studies following the 1997H5N1 outbreak in Hong Kong have shown that mildly symp-

tomatic or asymptomatic infections had occurred in a few in-dividuals exposed to infected patients or poultry (15, 16, 98).Similar studies of persons at risk for H5N1 exposure during therecent H5N1 outbreaks have shown little or no evidence ofhuman-to-human transmission in unprotected health careworkers exposed to H5N1 patients (129, 189). Similarly, villag-ers, poultry workers, and poultry cullers in Vietnam, Thailand,

Indonesia, and Cambodia who are heavily exposed to infectedpoultry rarely have clinical or asymptomatic (serological) evi-dence of infection (244). In contrast, around 10% of poultrystall holders in Hong Kong in 1997 had serological evidence ofH5N1 infection without presenting as overt H5N1 disease (15),although it is unclear whether the seropositivity representedrecent infection with HPAI H5N1 virus or prior infection byLPAI H5-subtype viruses known to be present in ducks. Al-though more serosurveillance data are needed to address thepossibility that the number of actual cases may be underesti-mated, the observations so far suggest the inefficient transmis-sion of current H5N1 viruses from infected poultry to humans.Therefore, the question is not why humans get infected with

H5N1 disease but why so many who are heavily exposed to the virus in areas where the virus is endemic fail to be infected,symptomatically or asymptomatically, by a virus that seems tobe ubiquitous (244). Conversely, while the affected cases inHong Kong in 1997 had significantly more exposure to live-poultry markets, approximately 30% of them had no obvioussource of infection (151). Similar observations were made elsewhere (97).

Among the human H5N1 cases, there is a significant numberof family clusters (8, 97, 165, 259). It is difficult to ascertainwhether these clusters represent infection from a commonenvironmental source or limited human-to-human transmis-sion. Excluding a common source of infections is epidemiolog-

ically exceedingly difficult, and only unusual circumstances al-low unequivocal proof of this (241). The lower case incidenceand lower case fatality rates for H5N1 in those over 40 years ofage remain unexplained (see above). Taken overall, it appearsthat while exposure to a source of H5N1 infection is necessary,such exposure alone is not sufficient to explain the observedepidemiology of H5N1 disease. Other as-yet-undeterminedfactors appear to be crucial in determining who gets infectedand ill. Among other possibilities, the role of host geneticsusceptibility factors and hitherto-unrecognized host resistancemechanisms (185) deserve investigation.

The Clinical Spectrum of Human H5N1 Infections

The age of cases ranged from 3 months to 75 years, with amedian age of 18 years (260). The first symptoms of influenzaH5N1 virus develop 2 to 4 days after the last exposure to sickpoultry, but longer incubation times of up to 8 days have beenreported. It is unknown whether and to what extent virus isshed during this time (8, 34, 97, 166, 232, 266). Most patientswith influenza H5N1 virus present with symptoms of fever,cough, and shortness of breath and radiological evidence ofpneumonia (8, 34, 232, 266). Abnormalities on chest radio-graphs are often bilateral and include diffuse, patchy, or inter-stitial infiltrates and segmental or lobular consolidation withair bronchograms. The pneumonia usually seems to be of pri-mary viral origin with no evidence of bacterial superinfection


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in most cases. This is supported by postmortem examinationsof a small number of patients (170, 230, 240). The propensityof current H5N1 viruses to cause lower respiratory tract infec-tions is in accord with the presence of avian-type SA -2,3 Galreceptors to which avian viruses can bind in human bronchiolarand alveolar cells (192, 243). Unlike human infections with H7viruses, conjunctivitis or upper respiratory symptoms do notseem to be prominent in H5N1-infected patients (34, 232, 266).Frequently occurring nonrespiratory symptoms include diar-rhea, vomiting, and abdominal pain. In some reported cases,diarrhea was the presenting symptom, preceding other clinicalmanifestations (3, 39).

Central nervous system (CNS) involvement in H5N1 influ-enza virus infection has been observed for one patient whoseillness was characterized by diarrhea, convulsions, and progres-sive coma and from whom H5N1 virus was isolated from ce-rebrospinal fluid and blood specimens (39). This patients sis-ter had also died of an undiagnosed illness of a similar nature2 weeks earlier, and this may suggest a possible genetic pre-disposition to this particular disease manifestation (39). Al-

though neurotropism of current H5N1 viruses has been notedin mammals such as mice, ferrets, and felids (103, 133, 180,224), CNS manifestations in human H5N1 disease seem to berare. It has been noted that seasonal influenza virus may alsorarely lead to CNS disease (150, 219). Fatal H5N1 disease hasbeen reported during pregnancy, but it is unclear (so far)whether transmission to the fetus has also occurred (198).

It should be noted that milder cases of H5N1 disease pre-senting as uncomplicated flu-like illness were reported in HongKong in 1997 (266) and in more recent outbreaks followingepidemiological investigations of index cases (97). Overall, inthe recent outbreaks, it is unclear whether the milder spectrumof human H5N1 disease is being missed through a lack of

adequate surveillance and diagnostic testing of mild cases.The clinical course of influenza H5N1 virus is often charac-terized by a rapid progression of lower respiratory tract dis-ease, necessitating mechanical ventilation within days of ad-mission to a hospital (8, 34, 232, 266). The median durationfrom the time of onset to hospitalization was 4 days, and thatfrom the time of onset to death for the fatal cases was 9 days(260). Progression to respiratory failure is frequently associ-ated with manifestations of ARDS. Other reported compli-cations include multiorgan failure with renal and cardiacdysfunction, Reyes syndrome, pneumothorax, pulmonaryhemorrhage, and ventilator-associated pneumonia (8, 34,232, 266).

Laboratory results characteristically seen in severe cases ofH5N1 disease include lymphopenia, often with an invertedratio of CD4-positive lymphocytes to CD8-positive lympho-cytes, thrombocytopenia and increased serum levels of livertransaminases, and, in some cases, hypoalbuminemia and ele-vation of lactate dehydrogenase and creatine kinase levels (8,34, 97, 166, 232, 266). High serum levels of cytokines andchemokines have been observed in several H5N1-infected pa-tients, suggesting a role of cytokine dysregulation in the patho-genesis of H5N1 disease (see below) (40, 170, 230). In mostcases, the immediate cause of death was respiratory failure.

Epidemiological analysis of the 256 confirmed H5N1 casesreported to the WHO between November 2003 and November2006 (260) revealed that over half of all cases occurred under

in patients under the age of 20 years, and 89% of the patientswere under 40 years of age. This skew in age distribution is notexplainable by the population-age structure of the affectedcountries (201). Based on reported cases, the mortality ofhuman influenza H5N1 virus was 60% (260). While this casefatality rate may be exaggerated because of biased case detec-tion, it seems to be clear that the severity of human H5N1

disease is very different from that caused by seasonal influenzaviruses. In contrast to human influenza virus, case fatality ratesof H5N1 disease were highest in the 10- to 19-year age group(76%) and lowest in those over 50 years (40%), although thenumber of cases in the age group of 50 years or over was small(n 15) (260). This unusual age distribution of case incidenceand case fatality may reflect age-related patterns of exposureor risk behavior (e.g., close contact with sick poultry) or age-related host resistance.

Pathogenesis of Human Influenza H5N1 Virus

Human H5N1 disease is clinically and pathologically distinct

from seasonal human influenza virus caused by H3N2 or H1N1viruses (see above). An understanding of the pathogenesis ofhuman H5N1 disease may derive from three sources: the clin-ical findings, virology, and pathology of human H5N1 disease;relevant animal models; and studies of cell-virus interactions invitro or ex vivo. While viral dissemination may contribute tothe unusual disease presentation, the primary pathology thatcontributes to death in most patients is the rapidly progressingfulminant primary viral pneumonia that often progresses toARDS. The target cells for H5N1 replication in the respiratorytract are not fully defined, but alveolar pneumocytes and mac-rophages have been identified by immunohistochemistry inautopsies (240), virus binding studies (243), and ex vivo infec-

tion of lung fragment cultures (159). Since both H5N1 andhuman H1N1 influenza viruses can replicate in the alveolarepithelium as well as the nasopharyngeal epithelium (159), adifferential tropism of H5N1 virus within the respiratory tractis unlikely to be a key explanation for the unusual pathogenic-ity of H5N1 viruses.

Human H5N1 disease differs from that of human influenza virus in terms of the viral load kinetics, virus disseminationbeyond the respiratory tract, and induction of hypercytokine-mia (40). The clinical manifestations of influenza H5N1 virusincluding diarrhea, liver, and renal dysfunction, severe lym-phopenia, and reactive hemophagocytosis suggest pathology inmultiple organs. This may suggest a wider tissue tropism of thevirus or may be the manifestations of multiple-organ dysfunc-tion that is related to the systemic effects of a severe sepsis-like syndrome. For example, it has been reported that Kuppfercell-dependent hepatitis is not uncommon in conventionalhuman influenza virus in the absence of virus infection in theliver (172).

Compared to human influenza virus, patients with H5N1disease have detectable viral RNA in the respiratory tract fora longer period, presumably because of the lack of prior cross-reactive immunity (40, 96). Higher levels of viral RNA in thenasopharynx and detection of viral RNA in the serum wereadverse prognostic factors (40). Virus has been isolated fromthe plasma, indicating the potential for systemic dissemination(35, 40). The demonstration of H5N1 RNA in feces from


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patients and in limited autopsy studies, together with theprominent diarrheal presentation of some patients with H5N1disease, suggests that the virus very likely affects the gastroin-testinal tract either as part of the initial infection or throughsubsequent dissemination (8, 40, 240). While the limited post-mortem examinations reported so far revealed no evidence ofviral replication or viral pathology in organs other than lungsand intestines (170, 230, 240), more studies, especially duringthe acute stage of infection, are essential to confirm or excludethe possibility of infection at sites other than the respiratoryand gastrointestinal tracts.

While the mechanisms of pathogenesis of HPAI viruses suchas H5N1 virus infection in chicken are well defined and aredetermined largely by the multibasic amino acids in the HAconnecting peptide (see above) and the consequent broad tis-sue tropism of the virus, these findings cannot be directlyextrapolated to mammals or to human disease. H5N1 virusesinfect BALB/c mice without prior adaptation. Virulence ofH5N1 viruses in mice, ferrets, felids, and viverrids (Owstenscivets) is associated with virus dissemination beyond the respi-

ratory tract to involve multiple organs including the brain (26,58, 66, 76, 99, 100, 179, 180). However, primates experimen-tally infected with H5N1 virus do not manifest virus dissemi-nation, and pathology is restricted to the respiratory tract (177,178). Animal models differ among each other and from hu-mans with regard to the attachment of H5N1 virus to respira-tory tissues. Fluorescently labeled H5N1 viruses bound moreefficiently to the alveolar epithelium than tracheal epitheliumin humans, ferrets, cats, and macaques, but the reverse wastrue in mice. Furthermore, while H5N1 virus attached to type2 pneumocytes in human, cat, and ferret lungs, the virus boundpredominantly to type 1 pneumocytes in macaques (243).Therefore, while mice are a convenient animal model for some

purposes (e.g., vaccine-induced protection from virus chal-lenge), the pathogenesis of H5N1 disease in mice probablydiffers from that in humans in important ways.

Virus virulence and dissemination in the BALB/c mousemodel is determined by the amino acid replacements in thePB2 gene at position 627 (Lys) or 701 (Asn) and the presence ofa multibasic amino acid motif in the HA connecting peptide(76, 128). H5N1 viruses also exhibit various levels of virulencein ferrets (66, 143, 144, 269), although this variation is notdetermined predominantly by PB2 Lys627 or the HA connect-ing peptide (66, 144). Some virus isolates from human H5N1disease in Vietnam had the mutation PB2 Lys627, but no avianisolates in Vietnam examined so far did so (203). However, thismutation does not appear to correlate with clinical outcome inhumans (40). Conversely, while avian H5N1 viruses of theQinghai-like lineage have PB2 Lys627 (29), human infectionswith these viruses do not appear to be necessarily more viru-lent for humans (166). Interestingly, the same change (PB2Lys627) was also found in an H7N7 virus isolated from aninfected human who died of pneumonia during an HPAI virusoutbreak in poultry in The Netherlands in 2003, while virusesisolated from mild human cases and from infected chickenscontained Glu627 in PB2 (54). As the majority of avian viruseshave PB2 Glu627 (the exception being the Qinghai lineage), itis conceivable that the PB2 Lys627 mutation may be one ad-aptation of the virus to the mammalian host. However, recentstudies suggest that other amino acid changes in PB2, as well as

changes in the other two subunits of the polymerase complex,PB1 and PA, also play a role in mammalian adaptation andvirulence of HPAI viruses (55, 128, 184). Interestingly, the virulence of the reconstructed 1918 Spanish flu was also de-pendent on the whole polymerase gene complex rather than onindividual mutations (235) and was attributed to enhancedvirus replication competence. However, the replication com-

petence of HPAI H5N1 viruses was comparable to that ofhuman H1N1 viruses in primary human respiratory epithelialcells and macrophages (25, 32).

Occasionally, patients have presented with encephalitic dis-ease, with evidence of virus isolation from the cerebrospinalfluid clearly demonstrating dissemination to the CNS (seeabove) (39). The question of whether H5N1 virus is neuro-tropic in humans is of fundamental importance in the choice ofrelevant animal models to investigate drug treatment and alsoto study pathogenesis. However, the majority of patients donot have encephalitic manifestations as a major component oftheir illness.

Patients with H5N1 disease have higher serum levels of

proinflammatory cytokines and chemokines. Levels of macro-phage attractant chemokines CXCL10 (IP-10), CXCL9 (Mig),and CCL-2 (monocyte chemoattractant protein 1) and of neu-trophil attractant interleukin-8 (IL-8) were elevated to higherlevels in plasma of patients with H5N1 disease than in plasmaof patients with conventional human influenza virus and weresignificantly higher in H5N1 patients who died than in thosewho recovered (40, 170). IL-8 is believed to play a role in thedevelopment of ARDS (94). Furthermore, IL-10, IL-6, andgamma interferon levels were also more elevated in H5N1disease (40). Gamma interferon is known to be a strong in-ducer of CXCL10 and CXCL9. The elevation of plasma cyto-kine levels was positively correlated with pharyngeal viral load

(40) and may simply reflect more extensive viral replicationand consequent direct viral pathology rather than being caus-ative of the pathology observed in H5N1-infected patients. It isnotable, however, that the gene expression profiles induced inprimary human macrophages and primary human respiratoryepithelial cells infected in vitro by comparable infecting dosesof H5N1 and human influenza H1N1 (or H3N2) viruses areremarkably different. Compared with human H1N1 or H3N2virus, it was found that H5N1 viruses hyperinduce a range ofcytokines including tumor necrosis factor alpha, alpha and betainterferon, IL-1, CCL2, CCL3, CCL4, CCL5, and CXCL10from macrophages (32, 69). Similarly, H5N1 viruses differen-tially hyperinduced CXCL10, IL-8, IL-6, CCL2, beta inter-feron, and CCL5 from primary lung pneumocytes (25). Thus,many of the cytokines that were found to be elevated in thesera of patients with H5N1 disease were differentially up-reg-ulated by the H5N1 virus in vitro, suggesting that the increasedlevels of cytokines in peripheral blood of patients with H5N1disease may be mediated by the ability of H5N1 viruses tohyperinduce cytokine cascades. Thus, the elevated levels ofcytokines and chemokines in human H5N1 disease may be acause rather than a consequence of the severe pathology.

The NS1 protein is an NS protein that is known to play a rolein modulating the host innate immune responses (59). Whilethe H5N1-mediated cytokine hyperinduction in macrophageswas mediated partly by the H5N1 virus NS gene segment (32),other gene segments also play an important role (J. S. M.


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Peiris, unpublished data). Studies in vivo of mice infected witha recombinant H1N1 virus possessing the 1997 H5N1 NS genecaused a cytokine imbalance in the lungs characterized byincreased concentrations of proinflammatory cytokines and de-creased levels of anti-inflammatory cytokines (132). In humaninfluenza virus, the NS1 gene or its product contributes to viralvirulence by allowing the virus to evade activating an interferonresponse in the host (59, 60, 114). Recombinant H1N1 virusescontaining the NS gene of 1997 H5N1 viruses were morepathogenic in pigs, possibly by escaping the antiviral effects ofinterferons and tumor necrosis factor alpha (190). This en-hanced virulence in pigs required the presence of glutamic acidinstead of aspartate at position 92 (Glu92) of the H5N1 NSgene, but this amino acid change has not been found in re-cently discovered H5N1 viruses isolated from humans (2, 127,203). In addition, the postsynaptic density protein-95, disc-large tumor suppressor protein, zonula occludes-1 (PDZ) li-gand motif found in carboxy terminus of the NS1 of avianinfluenza virus has been identified as a potential virus virulencefactor (162). The PDZ ligand binding motifs of NS1 of highly

pathogenic H5N1 viruses isolated in 1997 and 2003 as well asthe 1918 pandemic virus (all of avian origin) are able to bindcellular PDZ-containing proteins. However, NS1 of most low-pathogenicity human influenza viruses contain no such bindingproperties. Binding of viral NS1 to PDZ domain-containingproteins may disrupt PDZ domain protein-protein interactionsand affect cell pathways including those that regulate cell sig-naling, protein trafficking, and maintaining cell polarity andorganization.

Autopsy studies revealed that patients dying of H5N1 dis-ease had massive macrophage infiltrates in the lung, which canbe explained by the induction of macrophage-tropic chemo-kines (see above). Furthermore, those patients had evidence of

reactive hemophagocytic syndrome, which is believed to be acytokine-driven condition (170, 230).The severe lymphopenia observed in patients and in animal

models with H5N1 infection may be secondary to virus-in-duced apoptosis, as suggested by in vitro and murine experi-ments with H5N1 viruses (237, 267). Macrophages infected invitro with H5N1 viruses differentially hyperinduce tumor ne-crosis factor-related apoptosis-inducing ligand and cause theapoptosis of cocultured lymphocytic cell lines (267). An alter-native explanation for H5N1-associated lymphopenia may bethe suppression of hematopoiesis in the bone marrow.

It is interesting that, in comparison to contemporary humanH1N1 viruses, the reconstructed 1918 pandemic H1N1 viruswas also reported to differentially activate host gene expressionprofiles in lungs of experimentally infected mice (223) andmacaques (110). As with H5N1 viruses, the pathways differen-tially hyperinduced by the 1918 H1N1 virus in mouse lung werethose associated with cytokine induction and apoptosis. This isparticularly relevant because the clinical features of humanH5N1 disease shares some features with the 1918 H1N1 pan-demic, viz., a rapidly progressive primary viral pneumonia (insome cases, at least) and a higher case fatality rate in healthyadolescents and young adults. On the other hand, the geneexpression profile of the 1918 H1N1-infected macaques indi-cated a reduced sensitivity to type I interferons (110).

In summary, the pathogenesis of avian influenza H5N1 vi-ruses is likely to be multifactorial in nature, involving increased

viral replication competence, viral dissemination, differences intissue tropism, and differential gene expression responses in

infected host cells (Fig. 4). While some of these mechanismscan be blocked by efficient antiviral therapy, a better under-standing of the mechanisms underlying differential activationof host genes (123) may permit specifically directed immuno-modulation to contribute to therapy of this disease.

Laboratory Diagnosis

This section addresses issues pertaining to laboratory diag-nosis of human H5N1 disease and does not cover veterinarydiagnosis. In view of the nonspecific nature of the illness,laboratory confirmation of H5N1 influenza virus is essential.Laboratory confirmation of a diagnosis of H5N1 disease is,

however, challenging. It requires a high index of suspicion andthe most sensitive detection methods available (e.g., reversetranscriptase PCR [RT-PCR]) and may require the testing ofmultiple specimens (97, 166). The options for diagnosing in-fluenza virus in clinical specimens include virus culture, anti-gen detection, detection of viral nucleic acids by RT-PCR, anddetection of rising titers of antibodies. In the absence of epi-demiological links to areas with H5N1 influenza virus activity,further subtyping is not essential for routine diagnostics. How-ever, in countries where avian influenza H5N1 virus is knownto be active, patients with severe pneumonia of unexplainedetiology should be investigated virologically for influenza virusand, if positive, further investigated using H5-subtype-specificassays so that appropriate therapy, infection control measures,and timely epidemiological investigations can be initiated.Therefore, there is a need for rapid diagnostic assays whichdistinguish influenza virus subtypes.

Clinical specimens for virus detection. Virus has been iso-lated and viral RNA has been detected in respiratory speci-mens obtained from H5N1-infected patients for up to 16 daysafter the onset of illness, indicating that virus is shed and canbe detected for prolonged periods (8). Nasopharyngeal aspi-rates (NPA) and nasopharyngeal, throat, and nose swabs haveall been used for the detection of H5N1 virus, but it remainsunclear which is the diagnostic specimen of choice, becauseparallel studies comparing different diagnostic specimens arelimited. Nasal and pharyngeal swabs have been tested in par-

FIG. 4. Mechanisms of pathogenesis of human H5N1 disease.


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allel during recent outbreaks in southeast Asia, and this com-parison suggests higher virus loads and consequent higher di-agnostic yields in throat swabs than in nose swabs (8, 40, 97).NPA were successfully used for H5N1 diagnosis in Hong Kongduring the H5N1 outbreak in 1997 (266), but data directlycomparing diagnostic yields from NPA and pharyngeal swabswith other respiratory specimens are lacking. An advantage ofNPA is that it provides the ideal specimen for the rapid diag-nosis of many other respiratory virus infections (e.g., humaninfluenza A or B virus, adenovirus, and parainfluenza virus),which may help to exclude a diagnosis of H5N1 influenza virus,although dual infections with other respiratory viruses remaina possibility. Limited data suggest that viral load is higher inthe lower respiratory tract (e.g., endotracheal aspirates) thanin throat or nose swabs (40). Thus, where available, endotra-cheal aspirates or bronchoalveolar lavages are likely to repre-sent the optimal diagnostic specimens for the diagnosis ofH5N1 disease.

H5N1 virus has also been isolated and viral RNA has beendetected in feces and sera in some but not all H5N1 patients

tested and in the cerebrospinal fluid of one patient (8, 39, 40,240). However, for screening purposes, respiratory specimensremain the first choice. In H7N7-infected patients, conjunctivalswabs appeared to be the specimen of choice for virus detec-tion (54). However, there appears to be a significant differencein the tropisms of H7- and H5-subtype viruses for the humanconjunctiva, with conjunctivitis being a common manifestationin H7N7 infections but not in H5N1 infection (see above).There is no systematic data on the utility (or lack thereof) ofconjunctival swab specimens for the diagnosis of human H5N1disease. Autopsy specimens are critical for confirming or ex-cluding avian H5N1 influenza virus disease. If a full autopsy isnot possible, paramortem biopsies are alternative options.

Specimens should be transported on ice and tested freshupon receipt in the laboratory. For long-term storage of spec-imens for virus detection or isolation, they should be frozen at70C, ideally in multiple aliquots. Respiratory specimensshould be placed into virus transport medium. WHO guide-lines for specimen collection and laboratory testing for H5N1diagnosis are available (see http://www.who.int/csr/disease/avian_influenza/guidelines/labtests/en/index.html).

Virus isolation. H5N1 viruses can be isolated by inoculationof embryonated eggs or of Mardin-Darby canine kidney(MDCK) or other permissive cell lines. While culture of sea-sonal human influenza A viruses requires the addition of ex-ogenous trypsin for growth in MDCK cells, H5N1 virus andother HPAI viruses are not dependent on exogenous trypsinsupplements for growth. Virus culture still represents the goldstandard for diagnosis, and virus isolates are essential forfurther genetic and antigenic characterization of avian influ-enza viruses. However, because of the length of time requiredfor virus culture and the need for biosafety level 3 (BSL-3)laboratory facilities for culturing HPAI viruses, RT-PCRrather than virus isolation is usually the first diagnostic testapplied to suspected clinical specimens.

Antigen detection. Detection of viral antigens in clinicalspecimens by direct immunofluorescence and enzyme immu-noassay (EIA) is widely used for the diagnosis of human in-fluenza virus because of their rapidity. Presently, such testing isdirected at conserved viral antigens (e.g., nucleoprotein and

matrix protein) and does not differentiate human from avianinfluenza virus subtypes. The EIA-based methods are simpleand convenient to use and could theoretically be applicable aspoint-of-care tests. Commercially available antigen detectionEIA test kits have comparable analytical sensitivities for hu-man and avian influenza viruses, but their overall sensitivitywas 1,000-fold lower than that for virus isolation (24). Thus,

currently, viral antigen detection tests, while having acceptableclinical sensitivity for the diagnosis of human influenza viruses,appear to have low clinical sensitivity for the diagnosis of avianinfluenza H5N1 virus (8, 97, 166, 170, 266). Aside from thisapparently poor clinical sensitivity, a positive antigen test onlyconfirms a diagnosis of influenza A virus. Thus, it would re-quire additional subtype-specific diagnostic methods (e.g., RT-PCR or culture) to differentiate avian from human influenzavirus. Although H5-subtype-specific antigen detection tests arenow becoming available on an experimental basis and areundergoing evaluation for the diagnosis of diseased poultry,the current commercially available antigen detection testsseem to have limited clinical utility for the diagnosis of H5N1

disease in humans.RT-PCR. RT-PCR assays need to be targeted at genes (e.g.,

matrix gene) that are relatively conserved in order to detect allinfluenza A viruses and, separately, at the HA or NA genes toidentify specific influenza A virus subtypes. Usually, a panel ofsuch RT-PCR assays, which includes generic influenza A virusdetection plus specific detection of H5, H3, and H1 subtypes,is used to investigate suspected human H5N1 disease. Thisstrategy helps overcome potentially false-negative PCR resultsdue to the mutation of the HA gene because a specimen witha positive matrix gene that is negative for H5, H3, and H1would flag that specimen for more detailed investigation. In-cluding the time needed for viral RNA extraction and analysis

of the amplification products, the turnaround time for conven-tional RT-PCR assays is 6 to 8 h (or typically overnight). Theuse of real-time PCR shortens the turnaround time to around4 to 6 h, increases sensitivity and specificity by the use ofprobes, and enables the quantitation of the viral target gene(39, 157). Even more importantly, because these are closedsystems, the risk of PCR cross-contamination is minimized.The existence of several distinct sublineages and the high mu-tability of H5N1 viruses (28, 29, 202) pose a challenge formolecular diagnostics and necessitate continued evaluation,and possibly the modification of primers or probes, over time.Alternative molecular detection methods such as loop-medi-ated isothermal amplification tests have also been used, al-though they are not in routine use (88, 173).

Antibody detection. The detection of H5N1-specific antibod-ies is essential for epidemiological investigations. Because ofthe delayed seroconversion and the need for paired sera, se-rology can provide retrospective confirmation of H5N1 infec-tion. While HI is the preferred method for the detection ofsubtype-specific antibodies to human seasonal influenza vi-ruses in human sera, conventional HI tests (using avian orhuman erythrocytes) have limited value for detecting antibod-ies against avian viruses in humans and other mammals be-cause of low sensitivity (7, 104, 136, 182). Comparison of HIantibody tests with detection of neutralizing antibodies inH5N1-infected persons from the 1997 Hong Kong outbreakshowed the latter to be more sensitive (182). Based on these


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observations, neutralization assays have become the methodsof choice for the detection of H5-specific antibodies in hu-mans. Using these assays, antibodies against H5N1 virus weregenerally detected 14 or more days after the onset of symptomsin patients infected during the 1997 Hong Kong outbreak (98).This is comparable to kinetics of the antibody response duringprimary infection with human influenza viruses.

While ne

threat to human

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